Parathyroid hormone (referred to as PTH hereinafter) is a peptide hormone consisting of 84 amino acid residues, which is responsible for bone and calcium metabolism. A peptide fragment consisting of the first to 34th amino acid residues at the N-terminal of PTH, which is called PTH (1-34), has the same biological activity as PTH. On the other hand, other peptide fragments which lack the first few N-terminal amino acid residues, PTH (3-34), PTH (7-34), and the like, are known to suppress PTH activity.
Recently, it has been found that a PTH-related peptide (referred to as PTHrP hereinafter) derived from human carcinoma exhibits biological activity similar to PTH, and its chemical structure has been determined (Suva et al, Science, Vol.237, 893, 1987). The human PTHrP is a polypeptide consisting of 141 amino acid residues. It has biological activity similar to that of PTH, such as elevation of blood calcium level, acceleration of born absorption, lowering of blood phosphorous level, lowering of urinary calcium level, increasing of urinary cAMP level, and activation of hydroxylase at the 1-position of vitamin D in kidney (Horiuchi et al, Science, Vol.238, 1988; Kemp et al, Science, Vol.238, 1988).
Primary structure of PTHrP has poor similarity to that of PTH although partial structure of PTHrP at the amino terminal shows similarity to that of PTH. In spite of the fact, fragments of PTHrP, which lack a few amino terminal residues, such as PTHrP (3-34), suppress PTH activity likewise in PTH (Rabbani et al, oral speech at the meeting of the America Bone Metablism Association, 1988).
PTH derivatives such as [Tyr.sup.34 ]-hPTH (3-34)-NH.sub.2 and PTHrP derivatives such as hPTHrP (3-34)-NH.sub.2 are known as a PTH antagonist. However, there has been a need to discover move potent PTH antagonist activity. The present invention relates to the peptides consisting of 25-50 amino acid residues and comprising the following peptide sequence, and amides and salts thereof. EQU Leu-Met-His-Asn-Leu-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His -His-Leu-Ile-Ala-Glu-Ile (Seq Id No. 1) (I)
The following abbreviations are used in this text.
Asp: aspartic acid PA0 Thr: threonine PA0 Ser: serine PA0 Asn: asparagine PA0 Gln: glutamine PA0 Glu: glutamic acid PA0 Gly: glycine PA0 Ala: alanine PA0 Met: methionine PA0 Ile: isoleucine PA0 Leu: leucine PA0 Phe: phenylalanine PA0 Lys: lysine PA0 His: histidine PA0 Arg: arginine PA0 Boc: t-butoxycarbonyl PA0 Z: benzyloxycarbonyl PA0 OcHx: cyclohexyl ester PA0 OBzl: benzyl ester PA0 Bzl: benzyl PA0 Tos: p-toluenesulfonyl PA0 Cl-Z: 2-chlorobenzyloxycarbonyl
The peptide derivatives of the present invention contain at least the peptide sequence represented by the formula (I) mentioned above. For instance, the peptide derivatives of the invention may be represented by the following formula (II): EQU X-Leu-Met-His-Asn-Leu-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-H is-His-Leu-Ile-Ala-Glu-Ile-Y-z (II)
wherein X represents H, (Seq. ID Nos: 2-5), H-Gln (Seq. ID Nos: 6-9), H-A-Gln, (Seq. ID Nos. 6-9) H-Glu-A-Gln- (Seq. Id. Nos: 14-17), or Ser-Glu-A-Gln- (Seq. ID Nos: 18-21), wherein A is Ile, Thr, Val, or Leu; Y represents His-Thr-Ala, His-Thr-Ala-Glu-Ile-Arg-Ala, His-Thr-Ala-Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val, His-Thr-Ala-Glue-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser-Lys-Pro-Asn; Z represents OH or NH.sub.2.
The following Table 1 lists specific examples of the peptide derivatives of the invention.
TABLE 1 __________________________________________________________________________ X--Leu--Met--His--Asn--Leu--Gly--Lys--Ser--Ile--Gln--Asp--Leu--Arg--Arg-- Arg--Phe--Phe--Leu--His--His--Leu--Ile--Ala--Glu--Ile--Y--Z Compound No. X Y Z __________________________________________________________________________ 1 H--Ser--Glu--Ile--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 18) 2 H--Glu--Ile--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 14) 3 H--Ile--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 10) 4 H--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 6) 5 H His--Thr--Ala NH.sub.2 (SEQ ID NO: 2) 6 H--Ser--Glu--Ile--Gln His--Thr--Ala OH (SEQ ID NO: 18) 7 H--Ser--Glu--Ile--Gln His--Thr--Ala--Glu-- NH.sub.2 (SEQ ID NO: 19) Ile--Arg--Ala 8 H--Ser--Glu--Ile--Gln His--Thr--Ala--Glu-- NH.sub.2 (SEQ ID NO:20) Ile--Arg--Ala--Thr-- Ser--Glu--Val 9 H--Ser--Glu--Ile--Gln His--Thr--Ala--Glu-- NH.sub.2 (SEQ ID NO: 21) Ile--Arg--Ala--Thr-- Ser--Glu--Val--Ser-- Pro--Asn--Ser--Lys-- Pro--Asn 10 H--Ser--Glu--Val--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 18) 11 H--Ser--Glu--Thr--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 18) 12 H--Ser--Glu--Leu--Gln His--Thr--Ala NH.sub.2 (SEQ ID NO: 18) __________________________________________________________________________
The peptide derivatives of the invention may be used after conversion to pharmacologically acceptable salts thereof, such as hydrochloride or acetate.
The PTHrP derivatives of the invention represented by the formula (I) or (II) may be prepared by repeating condensation reaction between relevant protected amino acids by means of conventional solid phase method, said reaction being sequentially conducted starting from C-terminal and according to the amino acid sequence shown in formula (I) or (II), and removing the protective groups and carrier to which the C-terminal amino acid residue has been linked by known methods such as acid decomposition and aminolysis. The peptide synthetic method mentioned above and the starting amino acid derivatives used therein are described in detail in various text books (See Izumiya et al, "Basis and Practice of Peptide Synthesis", published by Maruzen, 1985; Gross and Meienhofer's, "The Peptides", Vol.2, Academic Press, 1980).
The solid phase carriers used in the peptide synthesis for preparing the peptide derivatives of the present invnetion may be conventional ones, and specific examples are polystyrene resins of substituted benzyl type, polystyrene resins of hydroxymethylphenylacetic amide type, substituted benzhydrylpolystyrene resins or polyacrylamide resins having a functional group for binding to a peptide. Amino acids condensation may be also conventional, and dicyclohexylcarbodiimide (DDC), acid anhydride, and activated ester methods may be used.
Protective groups in the starting protected amino acids may be groups which are known in conventional peptide synthesis and easily removed by conventional means such as acid decomposition, reduction or aminolysis. Specific examples of amino protective group are formyl; trifluoroacetyl; benzyloxycarbonyl; substituted benzyloxycarbonyls such as (ortho- or para-) chlorobenzyloxycarbonyl, and (ortho- or para-) bromobenzyloxycarbonyl; and aliphatic oxycarbonyl such as t-butoxycarbonyl and t-amyloxycarbonyl. Carboxylic acid in amino acids may be protected by being converted to ester group. As the ester group, there may be mentioned benzyl ester; substituted benzyl ester such as methoxybenzyl ester; alkyl esters such as cyclohexyl ester, cycloheptyl ester, or t-butyl ester. Guanidino group does not require any protective group, but may be protected by nitro; or arylsulfonyl such as tosyl, methoxybenzenesulfonyl, or mesithylenesulfonyl. Protecting groups for imidazole include tosyl, benzyl, and dinitrophenyl. Hydroxy groups present in serine and threonine molecules may be non-protected or protected by benzyl or substituted benzyl. The indole group in tryptophan molecule may be non-protected or protected by formyl or the like.
The final deprotection and detachment of a resultant peptide from the carrier may be conducted by the action of anhydrous hydrogen floride in the presence of one of various scavengers. Examples of the scavengers are anisole, (ortho-, metha-, or para-) cresol, dimethylsulfide, thiocresol, ethanediol, and mercaptopyridine, which are all conventional in peptide synthesis. Purification of the resultant peptide may be conducted by means of conventional methods, such as gel-filtration, ion-exchange chromatography, and high- or low-pressure reverse phase chromatography. The peptide thus purified may be converted to its salt by the use of gel-chromatography equilibrated with aqueous acetic acid or aqueous hydrochloric acid.